Tyrosine kinase pathways play a critical role in many cellular functions, including immune responses, integrin activation, granule release, growth and differentiation. The crucial ITAM (immune tyrosine activation motif) containing platelet receptor involved in the activation of platelets by collagen is Glycoprotein VI (GPVI) which signals through the FcR? chain to Syk, LAT, and PLC?2. Platelets also express CLEC-2, which is a hemITAM receptor, with a single tyrosine in the intracellular domain that also activates Syk, LAT, and PLC?2. Our overall hypothesis is that the hemITAM and ITAM receptors activate distinct signaling pathways and are differentially regulated by intracellular signaling molecules. Whereas the signaling events involved in the GPVI (ITAM)-mediated Syk activation are well established, mechanisms involved in CLEC-2-mediated Syk activation are not clear. Aim 1. We hypothesize that Syk bound to the hemITAM exists in a conformation that is recognized by Tec kinases and is phosphorylated by specific tyrosine residues for its activation. In contrast, ITAM bound Syk can be activated without a need phosphorylation. We will test this hypothesis using pathway inhibitors, while simultaneously comparing the effects of these inhibitors on the ITAM receptor (GPVI) and the hemITAM receptor (CLEC-2) signaling and Syk phosphorylation. We will identify the specific phosphorylation site on Syk by Tec kinases that leads to Syk activation when it is bound to hemITAM using a) phospho-specific antibodies, b) molecular cell biological approaches, and c) signaling and functional analysis of Syk knock-in mice in which specific tyrosines are mutated to phenylalanine. Finally we will evaluate the implications of pathway blockade and knock-in mice on the physiological function of CLEC2, i.e. separation of circulating blood from the lymphatic and blood vessels., and in vivo thrombosis models We propose that the hemITAM receptor CLEC-2 utilizes PI3 kinase for the activation of Tec kinases, Btk and Tec, leading to the phosphorylation and activation of Syk. We will test this hypothesis using pathway inhibitors and platelets from knockout mice, while simultaneously comparing the CLEC-2-mediated signaling events to those downstream of the ITAM receptor GPVI. We have strong preliminary data to support distinct signaling mechanisms in the activation of Syk by ITAM and hemITAM receptors in platelets. We have identified that the hemITAM receptor CLEC-2 utilizes the SFK/PI3K/Tec pathway to activate Syk, while the ITAM receptor GPVI does not. Aim 2. We propose that CLEC-2 and GPVI signaling pathways are differentially regulated in platelets by different signaling molecules. We also have strong preliminary evidence for the regulation of hemITAM and ITAM receptors in platelets by distinct signaling molecules. We will evaluate the role of Fyn kinase, Lyn kinase TULA2 phosphatase, Cbl proteins, and two small G proteins, RRas2 and RhoG in the regulation of signaling events and platelet functional responses by CLEC2 and GPVI, using mice deficient in these signaling molecules. Our preliminary data with Lyn null murine platelets show that CLEC-2 signaling is abolished in these platelets, while GPVI signaling is potentiated. Based on these strong preliminary data we postulate that Lyn phosphorylates CLEC-2 hemITAM that enables it to bind PI3 kinase. The studies proposed in this application thus identify distinct pathways mediated by the hemITAM receptor CLEC2 and the ITAM receptor GPVI in platelets and shed new light on the novel regulation of these pathways. Understanding these signaling cascades in platelets will help us evaluate and anticipate possible implications of the therapeutic agents that could interfere with these pathways.